BCL11B regulates IL-2 promoter activity. (A-B) MSCV-BCL11B and MSCV Jurkat cells were electroporated with the -585 IL-2 promoter-Firefly luciferase and CMV-Renilla luciferase constructs. Twenty-four hours after transfection, the cells were treated with PMA/ionomycin for 8 hours (A) or grown for 8 hours in the presence of plate-bound anti-CD3 and soluble anti-CD28 antibodies (B). The luciferase activity was measured using the Promega luciferase dual system. (C) Jurkat cells were electroporated with pCDNA3::BCL11B or pCDNA3 vectors and -585 IL-2 promoter-Firefly luciferase and CMV-Renilla luciferase constructs. Luciferase activity was measured as in panels A and B. (D) Western blot analysis of BCL11B after transfection of Jurkat cells with BCL11B specific (lane 2) or nontargeting (lane 1) siRNAs. HDAC2 was used as loading control. (E-F) Jurkat cells were transfected with the -585 IL-2 promoter-Firefly luciferase, CMV-Renilla luciferase constructs, and BCL11B specific or nontargeting siRNAs. (E) Luciferase activity was measured after PMA/ionomycin treatment for 8 hours; control cells lacked PMA/ionomycin treatment. (F) Secretion of IL-2 was determined by ELISA in media after treatment with PMA/ionomycin for 16 hours. The quantifications represent means of 3 independent experiments ± SD.